This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Breast cancer is the most common form of cancer diagnosed in women worldwide, affecting an estimated 10% of women. More than half of the cases are in industrialized countries and while the rate of mortality as a result of breast cancer has decreased due to early detection, the incidence of breast cancer has risen by 30% in these countries in the last quarter of a century. Developing novel techniques for treating breast cancer should therefore be of primary interest and gene therapy offers a promising approach in that regard. Suicide-gene or gene-directed enzyme prodrug therapy (GDEPT) is a strategy developed to selectively target cancer cells. Here, treatment of cancer is based on the transfer of non-mammalian genes into tumor cells. These transgenes encode enzymes that selectively convert non-toxic prodrugs to highly toxic metabolites. For example, expression of the herpes simplex virus thymidine kinase (HSVtk) gene in mammalian cells renders these cells susceptible to the cytotoxic effects of innocuous nucleotide analogues such as ganciclovir (GCV) after phosphorylation by the viral enzyme. Treatment with HSVtk/GCV not only kills HSVtk expressing cells but also neighboring cells that do not express the HSVtk gene, greatly enhancing the efficacy of HSVtk-mediated cytotoxicity. Viral systems are the most efficient delivery systems;however, problems with immunogenicity, the potential for mutagenicity and the difficulty and expense of producing large amounts of pure virus hamper their successful application in a clinical setting. In contrast, non-viral systems have several advantages: they are easy and inexpensive to produce, are able to incorporate large transgenes and do no illicit an immune response. However, to date many non-viral gene therapy strategies have been limited by the efficiency of this gene delivery systems and the lack of stable chromosomal integration. Transposable elements (transposons) are mobile genetic elements that represent a novel non-viral DNA delivery system that provides long-term expression of transgenes as they are able to efficiently and permanently integrate into the host genome. PiggyBac (pB), a transposable element originally isolated from the cabbage looper moth Trichoplusia ni showed efficient transposition and long-term transgene expression in vitro in a variety of cells and in vivo in mouse experiments. Recently, ultrasound-targeted microbubble destruction (UTMD) has been proposed as safe and efficient means for gene delivery. Microbubbles are commonly used as contrast agents in ultrasound imaging and it has also been shown that microbubble enhanced ultrasound can alter cell membrane permeability for a short time, allowing extracellular macromolecules such as plasmid DNA to instantaneously enter cells without cytotoxicity. Temporal and spatial administration of the ultrasound allows for controlled delivery of genes to specific tissues or organs at precise time points. In this application we propose to evaluate the feasibility of using UTMD in combination with the piggyBac transposon system as a tool to enhance gene directed enzyme prodrug therapy in human breast adenocarcinoma cell xenografts.